This proposal will focus on two aspects of mRNA turnover in differentiating leukemia cells. The first concerns changes in mRNA stability and their possible relationship to changes in mRNA structure and involves the comparison of complementary DNAs in noninduced and differentiating leukemia cells. The second concerns a novel mechanism for mRNA synthesis which may play an important role in terminal differentiation and involves an RNA-dependent RNA polymerase. We observed this activity while studying the metabolism of mRNA in differentiating murine erythroleukemia cells. The kinetics of globin mRNA synthesis in these cells, its subcellular localization, size, distribution among different RNA fractions, and drug sensitivity can be most easily explained by postulating cytoplasmic RNA-dependent RNA synthesis. Our discovery of "minus" globin RNA strand in differentiating MEL cells supports this conclusion. The present proposal will focus on three aspects of RNA-dependent RNA synthesis in murine erythroleukemia cells. The first deals with the purification and characterization of the protein components required for RNA-dependent RNA synthesis and with the question whether essential components may have general cellular function besides their involvement in erythropoetic differentiation. The second involves a detailed study of the mechanism of RNA-dependent RNA synthesis, including the mechanism of initiation of minus-strand synthesis, of RNA chain elongation, of synthesis of the positive strand from a minus-strand template, and the factors involved in template recognition. Most of these studies will be carried out with purified components, but parallel investigations will utilize an in vivo system consisting of enucleated cells that have been selectively permeabilized to precursors of macromolecular synthesis. The third aspect concerns the biological significance of RNA-dependent RNA synthesis and addresses itself to the questions whether this process is unique to erythropoetic differentiation or plays a role in other types of differentiation or perhaps even in all eucaryotic cells and whether it applies to the amplification of only certain types of mRNA or to all mRNA species.